Effects of Short-Term Nitrogen Addition on Soil Fungal Community Increase with Nitrogen Addition Rate in an Alpine Steppe at the Source of Brahmaputra

The soil fungal community plays a crucial role in terrestrial decomposition and biogeochemical cycles. However, the responses of the soil fungal community to short-term nitrogen addition and its related dominant drivers still remain unclear. To address this gap, we conducted an experiment to explore how different levels of nitrogen addition (five levels: 0, 2.5, 5, 10, and 20 g N m−2 y−1) affected the soil fungal community in an alpine steppe at the source of Brahmaputra. Results showed that the reduced magnitudes of soil fungal species and phylogenetic α-diversity increased with the increasing nitrogen addition rate. Nitrogen addition significantly changed the community composition of species, and the dissimilarity of the soil fungal community increased with the increasing nitrogen addition rate, with a greater dissimilarity observed in the superficial soil (0–10 cm) compared to the subsurface soil (10–20 cm). Increases in the soil nitrogen availability were found to be the predominant factor in controlling the changes in the soil fungal community with the nitrogen addition gradient. Therefore, short-term nitrogen addition can still cause obvious changes in the soil fungal community in the alpine grassland at the source of Brahmaputra. We should not underestimate the potential influence of future nitrogen deposition on the soil fungal community in the high-altitude grassland of the Qinghai–Tibet Plateau. Adverse effects on the soil fungal community should be carefully considered when nitrogen fertilizer is used for ecosystem restoration of the alpine grassland of the Qinghai–Tibet Plateau.


Introduction
With dramatically rising nitrogen (N) deposition worldwide during the last hundred years, studies on the effects of N deposition/addition on terrestrial ecosystems are increasing [1][2][3][4][5]. Preceding studies have concentrated on the responses of plant variables (e.g., productivity, α-diversity, community composition, and root trait), soil conventional physicochemical variables (e.g., pH, soil organic carbon, C:N, net N-mineralization, and base cations), soil microbial community (e.g., diversity, community composition, and biomass), and soil carbon storage to experimental N addition or atmospheric N deposition [5][6][7][8][9][10][11][12][13]. As is well known, the belowground soil microbial community is a crucial determining factor of their ecological functions [14,15]. Soil fungi are a diverse group of creatures, which play vital roles in governing terrestrial decomposition and biogeochemical cycles [16]. While some previous research has reported the response of the soil fungal community to nitrogen deposition [17][18][19], current studies have explored this topic far less than that of the soil bacterial community [20]. In addition, previous studies still have to address some issues that require further research. First, the response of the soil fungal α-diversity and community composition to short-term nitrogen addition gradients remains controversial [20][21][22][23][24], site. The dominant species in this steppe was Potentilla bifurca, accompanied by Microula sikkimensis, etc. Plant roots were mainly distributed at the 0-20 cm depth. The soil organic carbon and pH values were 0.57-0.81% and 8. 27-8.47, respectively [37].
The detailed information about nitrogen adding had been described by Huang et. al [37] and is briefly summarized here. In August 2021, a homogenous steppe was selected for the nitrogen fertilization experiment using a one-way factor experimental design. The field experiment with five N addition rates (0, 2.5, 5, 10, and 20 g N m −2 y −1 of urea) was conducted, which were characterized by N0 (control), N2.5, N5, N10, and N20, respectively. Each treatment had five replicates, and a total of twenty-five 5 m × 5 m plots were arranged founded on a stochastic design. The distance between the two adjacent plots was about 3 m.

Vegetation Community Examination, Soil Sampling, and Analysis
In September 2021, we recorded the total coverage existing in a 0.5 m × 0.5 m quadrat in the middle of every plot, and the number of plant species, height, and coverage of every plant species [38]. After plant community investigation, we used an auger of 3.8 cm in diameter to collect soil at a depth of 0-10 cm and 10-20 cm from every quadrat. Five soil cores were collected and then mixed to take a composite soil sample. A 2.0 mm sieve was used to remove stones and roots in the compound soil samples. Some of the soil samples were used to measure the soil fungal OTUs, and some were used to measure the soil physicochemical properties, including pH, soil moisture (SM), ammonium nitrogen (NH 4 + -N), nitrate nitrogen (NO 3 − -N), available phosphorus (AP), soil organic carbon (SOC), total phosphorus (TP), total nitrogen (TN), and soil enzymes, including sucrase (SC), cellulase (CL), urease (UA), catalase (CT), and alkaline phosphatase (ALP). The rates of SOC to TN (C:N), SOC to TP (C:P), TN to TP (N:P), NH 4 + -N to NO 3 − -N (NH 4 + -N:NO 3 − -N), and the sum of NH 4 + -N and NO 3 − -N to available phosphorus (AN:AP) were then calculated. We described the high-throughput sequencing procedures in detail in the Supplementary Materials. Analysis revealed 2267 OTUs in the surface soil and 2470 OTUs in the subsurface soil of the soil fungal community across the 50 samples.

Statistical Analysis
The response ratio was calculated to obtain the effect size of nitrogen addition for a particular variable [7,39].
where Ni was the nitrogen addition rate and N0 was the control treatment. R OTUs : effect size of nitrogen addition on operational taxonomic units of soil fungal community; R ACE : effect size of nitrogen addition on ACE index of soil fungal community; R Chao1 : effect size of nitrogen addition on Chao1 index of soil fungal community; R Shannon : effect size of nitrogen addition on Shannon index of soil fungal community; R Simpson : effect size of nitrogen addition on Simpson index of soil fungal community; R PD : effect size of nitrogen addition on phylogenetic diversity of soil fungal community; R MPD : effect size of nitrogen addition on mean pairwise distance of soil fungal community; R MNTD : effect size of nitrogen addition on mean nearest taxon distance of soil fungal community; R NRI : effect size of nitrogen addition on net relatedness index of soil fungal community; R NTI : effect size of nitrogen addition on nearest taxon index of soil fungal community.
R Shannonplant : effect size of nitrogen addition on Shannon index of plant community; R Simpsonplant : effect size of nitrogen addition on Simpson index of plant community; R SRplant : effect size of nitrogen addition on species richness of plant community; R Pielouplant : effect size of nitrogen addition on Pielou index of plant community; R SM : effect size of nitrogen addition on soil moisture; R AP : effect size of nitrogen addition on available phosphorus; R NH4+-N : effect size of nitrogen addition on ammonium nitrogen; R NO3−-N : effect size of nitrogen addition on nitrate nitrogen; R SOC : effect size of nitrogen addition on soil organic carbon; R TP : effect size of nitrogen addition on total phosphorus; R TN : effect size of nitrogen addition on total nitrogen; R C:N : effect size of nitrogen addition on ratio of carbon to nitrogen; R C:P : effect size of nitrogen addition on ratio of carbon to phosphorus; R N:P : effect size of nitrogen addition on ratio of nitrogen to phosphorus; R NH4+-N:NO3−-N : effect size of nitrogen addition on ratio of ammonium nitrogen to nitrate nitrogen; R AN:AP : effect size of nitrogen addition on ratio of available nitrogen to available phosphorus; R SC : effect size of nitrogen addition on soil sucrase; R CL : effect size of nitrogen addition on soil cellulase; R UA : effect size of nitrogen addition on soil urease; R CT : effect size of nitrogen addition on soil catalase; and R ALP : effect size of nitrogen addition on soil alkaline phosphatase.
We used the Microeco package to obtain the soil fungal species α-diversity (OTUs, ACE, Chao1, Shannon, and Simpson) and β-diversity [40]. ACE and Chao1 were proposed by Chao to estimate the number of OTUs in a community [41]. We used the Picante package to calculate the soil fungal phylogenetic α-diversity (PD, MPD, MNTD, NRI, and NTI) and β-diversity. We used the Microeco package to carry out a differential abundance test for soil fungal species based on the LEfSe method and then used Duncan multiple comparison to calculate the differential species of soil fungi. We used the Random Forest package to calculate the relative contribution to soil fungal species and phylogenetic αdiversity of every relevant variable [42]. We used the 'adonis2' function to test the soil fungal species and phylogenetic community composition between any two treatments with the nitrogen addition gradient. We used the Hmisc package to calculate the Pearson correlations between soil fungal species and phylogenetic α-diversity and soil and plant variables. We used the Microeco package to calculate the Mantel test between species and phylogenetic community composition of soil fungi and environmental variables. We used the iCAMP package to calculate the community assembly processes of the soil fungal community [29,43,44]. Software used in this study include R 4.0.2, SPSS 25, and Origin 2023.

Changes in Soil Fungal Species and Phylogenetic α-Diversity with the Nitrogen Addition Gradient and the Relation with Environmental Variables
The effect size of soil fungal species α-diversity, including OTUs, ACE, and Chao1 in surface soil and OTUs in subsurface soil, and soil fungal phylogenetic α-diversity, including PD, MPD, and NTI in surface soil and PD in subsurface soil, decreased with an increasing nitrogen addition rate (Figures 1a-d and 2a-d). However, the effect size of soil fungal species α-diversity, including Shannon, Simpson in surface soil and ACE, Chao1, Shannon, and Simpson in subsurface soil, and soil fungal phylogenetic α-diversity, including MNTD and NRI in surface soil and MPD, NTI, MNTD, and NRI in subsurface soil, showed no relationships with the nitrogen addition rate (Figures 1e,f, 2e,f, S1 and S2).      Figure S3a). The environmental variations included R SM , R CL , R ALP , R TN , etc., and they commonly explicated 91.32% variation of the R Simpson in surface soil with the nitrogen addition gradient ( Figure S3b). Further study found that R SM , R NO3−-N , etc., were dominant factors leading to significant changes in soil fungal species α-diversity, including R OTUs , R ACE , and R Chao1 in surface soil.
The environmental variations included R CT , R pH , R NO3−-N , R SM , R SRplant , R C:N , R TP , etc., and they commonly explicated 68.12% variation of the R OTUs in subsurface soil with the N addition gradient (Figure 3d). The environmental variations included R pH , R CL , R ALP , R CT , R NO3−-N , etc., and they commonly explicated 75.12% variation of the R ACE in subsurface soil with the N addition gradient (Figure 3e). The environmental variations included R CT , R CL , R pH , etc., and they commonly explicated 73% variation of the R Chao1 in subsurface soil with the N addition gradient; in particular, the predominant factor R CT had the most relative contribution (Figure 3f). The environmental variations included R UA , R AP , R pH , R NH4+-N:NO3−-N , R SC , R NH4+-N , etc., and they commonly explicated 65.94% variation of the R Shannon in subsurface soil with the N addition gradient ( Figure S3c). The environmental variations included R AP , R UA , R CL , R pH , R SOC , etc., and they commonly explicated 91.88% variation of the R Simpson in subsurface soil with the N addition gradient ( Figure S3d). Further study still found that R CT , R pH , R NO3−-N , etc., were the leading factors resulting in significant change in soil fungal species α-diversity, including R OTUs in subsurface soil. Microorganisms 2023, 11, x FOR PEER REVIEW 8 of 17  Figure S4c). The environmental variations included RpH, RTP, RALP, RPielouplant, RSM, etc., and they commonly explicated 58.05% variation of the RNRI in subsurface soil with the nitrogen addition gradient ( Figure  S4d). Further study also found that RCT, RpH, RNO3−-N, etc., were the dominant factors The environmental variations included R SM , R TP , R NO3−-N , R NH4+-N:NO3−-N , R CL , R CT, etc., and they commonly explicated 60.12% variation of the R PD in surface soil with the nitrogen addition gradient (Figure 4a). The environmental variations included R C:P , R TN , R CL , R NO3−-N , etc., and they commonly explicated 74.54% variation of the R MPD in surface soil with the nitrogen addition gradient (Figure 4b). The environmental variations included R TN , R AP , R NH4+-N:NO3−-N , R CL , R C:N , R TP , etc., and they commonly explicated 43.19% variation of the R NTI in surface soil with the nitrogen addition gradient (Figure 4c). The environmental variations included R Shannonplant , R CL , R AP , R SC , R C:P , R N:P , R pH , etc., and they commonly explicated 42.68% variation of the R MNTD in surface soil with the nitrogen addition gradient ( Figure S4a). The environmental variations included R SOC , R C:P , R UA , R Shannonplant , R pH , etc., and they commonly explicated 63.70% variation of the R NRI in surface soil with the nitrogen addition gradient ( Figure S4b). Further research also found that R SM , R NO3−-N , R NH4+-N:NO3−-N , etc., were the dominant factors that caused significant changes in the soil fungal phylogenetic α-diversity, including R PD , R MPD , and R NTI in surface soil. leading to significant changes in the soil fungal phylogenetic α-diversity, including RPD in subsurface soil.

Changes in Soil Fungal Species and Phylogenetic Community Composition with the Nitrogen Addition Gradient and the Relation with Environmental Variables
The soil fungal species β-diversity between the control and nitrogen addition treatments significantly increased with the increasing nitrogen addition rate in surface soil (Figure 5a, p < 0.01) but not in subsurface soil (Figure 5b). For the soil fungal species community, the Adonis2 texts showed that nitrogen addition treatments significantly changed the community composition (p = 0.02) and N0 treatment was extremely divergent to the N20 treatment in surface soil, while not in subsurface soil (Table 1). There were 10 diverse clusters of soil fungal species community in surface soil whose LDA points were >2, The environmental variations included R CT, R pH , R NO3−-N , R TP , R C:N , etc., and they commonly explicated 69.15% variation of the R PD in subsurface soil with the nitrogen addition gradient (Figure 4d). The environmental variations included R UA , R SC , R pH , R SOC , etc., and they commonly explicated 68.32% variation of the R MPD in subsurface soil with the nitrogen addition gradient (Figure 4e). The environmental variations included R pH , R UA , R SOC , R C:P , R N:P , etc., and they commonly explicated 49.70% variation of the R NTI in subsurface soil with the nitrogen addition gradient (Figure 4f). The environmental variations included R UA , R CT , R pH , R TP , etc., and they commonly explicated 70.28% variation of the R MNTD in subsurface soil with the nitrogen addition gradient ( Figure S4c). The environmental variations included R pH , R TP , R ALP , R Pielouplant , R SM , etc., and they commonly explicated 58.05% variation of the R NRI in subsurface soil with the nitrogen addition gradient ( Figure S4d). Further study also found that R CT, R pH , R NO3−-N , etc., were the dominant factors leading to significant changes in the soil fungal phylogenetic α-diversity, including R PD in subsurface soil.

Changes in Soil Fungal Species and Phylogenetic Community Composition with the Nitrogen Addition Gradient and the Relation with Environmental Variables
The soil fungal species β-diversity between the control and nitrogen addition treatments significantly increased with the increasing nitrogen addition rate in surface soil (Figure 5a, p < 0.01) but not in subsurface soil (Figure 5b). For the soil fungal species community, the Adonis2 texts showed that nitrogen addition treatments significantly changed the community composition (p = 0.02) and N0 treatment was extremely divergent to the N20 treatment in surface soil, while not in subsurface soil (Table 1). There were 10 diverse clusters of soil fungal species community in surface soil whose LDA points were >2, including one class, two orders, two families, three genera, and two species ( Figure  S5). There were eight diverse clusters of soil fungal species community in subsurface soil whose LDA points were >2, including one phylum, two orders, four families, and one genus ( Figure S6). Duncan multicomparison showed that o__Xylariales under control treatment was significantly higher than that under N20 treatment in surface soil ( Figure S5c). Duncan multicomparison also showed that g__Vishniacozyma under control treatment was significantly higher than that under others' treatments in surface soil ( Figure S5g). including one class, two orders, two families, three genera, and two species ( Figure S5). There were eight diverse clusters of soil fungal species community in subsurface soil whose LDA points were >2, including one phylum, two orders, four families, and one genus ( Figure S6). Duncan multicomparison showed that o__Xylariales under control treatment was significantly higher than that under N20 treatment in surface soil ( Figure  S5c). Duncan multicomparison also showed that g__Vishniacozyma under control treatment was significantly higher than that under others' treatments in surface soil ( Figure  S5g).

Figure 5.
Relationships between nitrogen addition rate and the effect size of nitrogen addition on soil fungal species β-diversity in surface soil (a) and subsurface soil (b) in an alpine steppe.  The soil fungal phylogenetic β-diversity showed no relationship with the nitrogen addition rate ( Figure S7). For the soil fungal phylogenetic community, the Adonis2 texts showed that N0 treatment was extremely divergent to the N20 treatment in the surface soil but not in the subsurface soil (Table 1).
According to the results of the Mantel test, the soil fungal species community composition was significantly correlated with NO 3 − -N, NH 4 + -N, and SC in the surface soil. The soil fungal species community composition was significantly correlated with UA, SC, and TN in the subsurface soil (Table 2). Note: "+", "*", and "**" means p < 0.1, p < 0.05, and p < 0.01, respectively.
The soil fungal phylogenetic community composition was significantly correlated with SC and marginally correlated with SM, NO 3 − -N, NH 4 + -N, and AN:AP in the surface soil, respectively. The soil fungal phylogenetic community composition was marginally correlated with Shannon plant , TN, SC, and UA in the subsurface soil, respectively (Table 2).

Effect of Nitrogen Addition on Community Assembly of Soil Fungal Community
The change in the effect size of nitrogen addition on the ecological processes in both surface and subsurface soil is shown in Figures 6 and S8. The 'Dispersal Limitation' process increased with the increasing nitrogen addition rate in surface soil (Figure 6a, p = 0.05), while others showed no relationships with the nitrogen addition rate (Figures 6b and S8).

Changes in Species and Phylogenetic α-Diversity with the Nitrogen Addition Gradient
We discovered that the reduced magnitude of soil fungal species and phylogenetic α-diversity enlarged with the increasing nitrogen addition rate, which may be due to several reasons. First, the soil nitrogen availability is an important factor influencing the species and phylogenetic α-diversity of the soil fungal community [45,46]. The effect size of nitrogen addition on soil NO3 − -N enlarged with the increasing nitrogen addition rate (Figure S9a,b) but was negatively interrelated with the species and phylogenetic α-diversity of the soil fungal community (Tables S1 and S2). Second, the pH is another main reason influencing the species and phylogenetic α-diversity of the soil fungal community [46][47][48][49]. The effect size of nitrogen addition on the soil pH was positively interrelated with the species and phylogenetic α-diversity of the soil fungal community (Tables S1 and S2). However, the effect size of the soil pH showed no relationship with the nitrogen addition rate (Figure S9c), and no significant change in soil pH among the five nitrogen addition treatments was observed [37]. Third, the soil moisture is also a chief reason influencing the α-diversity of the soil fungal community [50,51]. The effect size of nitrogen addition on soil moisture enlarged with the increasing nitrogen addition rate ( Figure S9d) but was negatively interrelated with the α-diversity of the soil fungal community (Tables S1 and  S2). Fourth, the plant α-diversity may also be related to the α-diversity of the soil fungal community [52,53], but there were no significant changes in the plant α-diversity along the nitrogen addition gradient [37]. Fifth, the soil fungal community α-diversity has no correlation with the soil enzyme activity [54], and no significant changes in SC, CL, UA, CT, and ALP were observed among the five nitrogen addition treatments [37]. Sixth, the relative contributions of AP, NH4 + -N, SOC, TP, TN, C:N, C:P, N:P, and AN:AP to the αdiversity of the soil fungal community were negligible.

Changes in Species and Phylogenetic Community Composition with the Nitrogen Addition Gradient
In this study, we found that short-term (<1 year) nitrogen addition treatments and a high nitrogen addition rate (20 g N m −2 y −1 ) could significantly change the soil fungal community composition in surface soil in the alpine steppe at the source of Brahmaputra. This discovery was in accordance with several studies that proved that long-term (5-53 years) nitrogen addition (8-32 g N m −2 y −1 ) could significantly change the soil fungal community

Changes in Species and Phylogenetic α-Diversity with the Nitrogen Addition Gradient
We discovered that the reduced magnitude of soil fungal species and phylogenetic α-diversity enlarged with the increasing nitrogen addition rate, which may be due to several reasons. First, the soil nitrogen availability is an important factor influencing the species and phylogenetic α-diversity of the soil fungal community [45,46]. The effect size of nitrogen addition on soil NO 3 − -N enlarged with the increasing nitrogen addition rate ( Figure S9a,b) but was negatively interrelated with the species and phylogenetic α-diversity of the soil fungal community (Tables S1 and S2). Second, the pH is another main reason influencing the species and phylogenetic α-diversity of the soil fungal community [46][47][48][49]. The effect size of nitrogen addition on the soil pH was positively interrelated with the species and phylogenetic α-diversity of the soil fungal community (Tables S1 and S2). However, the effect size of the soil pH showed no relationship with the nitrogen addition rate ( Figure S9c), and no significant change in soil pH among the five nitrogen addition treatments was observed [37]. Third, the soil moisture is also a chief reason influencing the α-diversity of the soil fungal community [50,51]. The effect size of nitrogen addition on soil moisture enlarged with the increasing nitrogen addition rate ( Figure S9d) but was negatively interrelated with the α-diversity of the soil fungal community (Tables S1 and S2). Fourth, the plant α-diversity may also be related to the α-diversity of the soil fungal community [52,53], but there were no significant changes in the plant α-diversity along the nitrogen addition gradient [37]. Fifth, the soil fungal community α-diversity has no correlation with the soil enzyme activity [54], and no significant changes in SC, CL, UA, CT, and ALP were observed among the five nitrogen addition treatments [37]. Sixth, the relative contributions of AP, NH 4 + -N, SOC, TP, TN, C:N, C:P, N:P, and AN:AP to the α-diversity of the soil fungal community were negligible.

Changes in Species and Phylogenetic Community Composition with the Nitrogen Addition Gradient
In this study, we found that short-term (<1 year) nitrogen addition treatments and a high nitrogen addition rate (20 g N m −2 y −1 ) could significantly change the soil fungal com-munity composition in surface soil in the alpine steppe at the source of Brahmaputra. This discovery was in accordance with several studies that proved that long-term (5-53 years) nitrogen addition  g N m −2 y −1 ) could significantly change the soil fungal community composition in grasslands or forests [5,18,23,55,56]. However, some studies reported that 2-3 years of nitrogen addition (10 g N m −2 y −1 ) did not significantly change the soil fungal community composition [22,34], which may be mainly due to the low nitrogen addition rate and short-term duration of nitrogen addition.
We found that the soil fungal β-diversity increased with the nitrogen addition rate in the surface soil, which may be due to several reasons. First, a previous study indicated that the available nitrogen is the key factor that influenced the soil fungal community composition [23]. In this study, the soil fungal community composition was significantly interrelated with NH 4 + -N and NO 3 − -N in surface soil (Table 2), and NH 4 + -N and NO 3 − -N significantly changed along the nitrogen addition gradient at the same time [37]. Second, the soil organic carbon is an important factor in determining the soil fungal community composition [57,58]. However, the soil organic carbon showed no significant changes with the increasing nitrogen addition rate [37]. Third, the total nitrogen, soil moisture, and soil enzyme activity have correlations with the soil fungal β-diversity [53,55,58], which did not significantly change across the nitrogen addition gradient [37]. Fourth, a previous study showed that the 'Dispersal Limitation' process results in relatively low similarity in the community [29]. In this study, the 'Dispersal Limitation' process increased with the nitrogen addition rate (Figure 6a).

Response Strength Is Stronger in Surface Soil than Subsurface Soil
In this research, the response strengths of the soil fungal community to nitrogen addition were different between soil depths, and the strength was stronger in surface soil than subsurface soil. There are a few reasons for this. First, it is well established that the soil fungal community differs between different soil depths due to different soil properties and community assembly processes in different soil depths [59,60]. Second, previous studies report that the enclosed microhabitat conditions in subsurface soil have been proven to be less affected by environmental changes than in surface soil [61,62]. Third, the soil fungal community is closely related to the available soil nitrogen, which varied in different soil depths due to nitrogen addition [63]. The predominant driving factors of soil NO 3 − -N had extremely significant differences between surface and subsurface soils.

Conclusions
Based on a short-term (<1 year) field nitrogen addition experiment in an alpine steppe at the source of Brahmaputra, we discovered that the negative effect of nitrogen addition on species and phylogenetic α-diversity of the soil fungal community and showed a decreased correlation with the nitrogen addition rate. Nitrogen addition significantly changed the species community composition of soil fungi, and the dissimilarity of soil fungal community caused by nitrogen addition increased with the increasing nitrogen addition rate. This phenomenon was stronger in surface soil than subsurface soil. Elevated nitrogen availability was the main driver of changes in soil fungal community. Therefore, the influences of nitrogen addition and the application of nitrogen fertilizer on the restoration of alpine steppes should be carefully reconsidered in the Brahmaputra River basin.
Supplementary Materials: The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/microorganisms11081880/s1, Figure S1. Relationships between nitrogen addition rate and the effect size of nitrogen addition on species α-diversity of soil fungi in an alpine steppe. Surface soil: (a) Shannon; (b) Simpson. Subsurface soil: (c) Shannon; (d) Simpson. Figure S2. Relationships between nitrogen addition rate and the effect size of nitrogen addition on phylogenetic α-diversity of soil fungi in an alpine steppe. Surface soil: (a) MNTD; (b) NRI. Subsurface soil: (c) MNTD; (d) NRI. Figure S3. Relative contribution of observed plant and soil characteristics to effect size of nitrogen addition on species α-diversity of soil fungi in an alpine steppe. Surface soil: (a) Shannon; (b) Simpson. Subsurface soil: (c) Shannon; (d) Simpson. Figure S4.
Relative contribution of observed plant and soil characteristics to effect size of nitrogen addition on phylogenetic α-diversity of soil fungi in an alpine steppe. Surface soil: (a) MNTD; (b) NRI. Subsurface soil: (c) MNTD; (d) NRI. Figure S5. Duncan multi-comparison of soil fungal community in surface soil at the class, order, family, genus, and species level along the N addition gradient based on LEFSe analysis. (Different letters indicate significant differences between nitrogen addition treatments). Figure S6. Duncan multi-comparison of soil fungal community in subsurface soil at the phylum, order, family, and genus level along the N addition gradient based on LEFSe analysis. (Different letters indicate significant differences between nitrogen addition treatments). Figure S7. Change in effect size of nitrogen addition on soil fungal phylogenetic β-MNTD in surface soil (a) and subsurface soil (b) along the N addition gradient in an alpine steppe. Figure Table S1. Pearson correlation analyses between species α-diversity and soil and plant variables. Table S2. Pearson correlation analyses between phylogenetic α-diversity and soil and plant variables.